The activation of T cells has been shown to require at least two signals via molecules present on professional antigen-presenting cells: signal 1 via a peptide/MHC complex, and signal 2 via a costimulatory molecule. Studies are being conducted on the impact of hyperexpression of multiple costimulatory molecules in the activation of T cells. Pox virus vectors (vaccinia and avipox) are being used because of their ability to efficiently express multiple transgenes. Murine tumor cells provided with signal 1 and infected with either recombinant vaccinia or avipox vectors containing a TRIad of COstimulatory Molecules (B7-1/ICAM-1/LFA-3, designated TRICOM) induce the activation of T cells to a far greater extent than cells infected with any one or two costimulatory molecules. Despite this T-cell "hyperstimulation" using TRICOM vectors, no evidence of increased apoptosis was seen. Studies have shown that dendritic cells infected with TRICOM vectors are rendered more efficient in enhancing T-cell responses. Peptide-pulsed dendritic cells infected with rF-TRICOM or rV-TRICOM induced cytotoxic T-lymphocyte activity in vitro and in vivo to a markedly greater extent than peptide-pulsed dendritic cells. These studies thus demonstrate the ability of vectors to introduce three costimulatory molecules into cells, thereby activating T-cell populations to levels far greater than those achieved with the use of one or two costimulatory molecules. This new threshold of T-cell activation in vaccine design is currently being studied.Local radiation is an established therapy for human tumors. Radiation also has now been shown to alter the phenotype of target tissue, including gene products that may make tumor cells more susceptible to T-cell-mediated immune attack. We have demonstrated a biological synergy between local radiation of tumor and active vaccine therapy. The model used consisted of mice transgenic for human carcinoembryonic antigen (CEA) and a murine carcinoma cell line transfected with CEA. The vaccine regimen consisted of a prime and boost strategy using vaccinia and avipox recombinants expressing CEA and three T-cell costimulatory molecules. One dose of 8-Gy radiation to tumor induced up-regulation of the death receptor Fas in situ for up to 11 days. However, neither radiation at this dose nor vaccine therapy was capable of inhibiting growth of established tumor. When vaccine therapy and local radiation of tumor were used in combination, dramatic and significant cures were achieved. This was mediated by the engagement of the Fas/Fas ligand pathway because Ag-bearing tumor cells expressing dominant-negative Fas were not susceptible to this combination therapy. Following the combination of vaccine and local radiation, tumors demonstrated a massive infiltration of T cells not seen with either modality alone. Mice cured of tumors demonstrated CD4(+) and CD8(+) T-cell responses specific for CEA but also revealed the induction of high levels of T-cell responses to two other antigens overexpressed in tumor, indicating the presence of a consequential antigen cascade. Thus, these studies demonstrate a new paradigm for the use of local tumor irradiation in combination with active specific vaccine therapy to elicit durable antitumor responses of established tumors.Intratumoral (i.t.) vaccination represents a potential modality for the therapy of tumors. Previous i.t. vaccination studies have focused on the efficacy of i.t. vaccination alone. There are no reports that clearly compared i.t. vaccination with systemic vaccination achieved by s.c., intradermal, or i.m. injection, or combining both modalities of systemic and i.t. vaccination. We have compared the antitumor effects induced by a systemic vaccination regimen (s.c.) and i.t. vaccination, and a sequential s.c/i.t. vaccination regimen. We have used a recombinant vaccinia virus containing the transgenes for carcinoembryonic antigen (CEA) and a triad of T-cell costimulatory molecules (designated rV-CEA/TRICOM) for s.c. priming and a replication defective avipox (fowlpox) virus containing the same four transgenes (designated rF-CEA/TRICOM) for i.t. vaccination or s.c. booster vaccinations. Vaccination was started on day 8 after s.c. implantation with CEA-positive tumors. We compared the antitumor activity induced by these vaccines when administered via the i.t. route versus the s.c. route. Subsequent therapy studies examined the sequential combination of these routes, s.c. priming with rV-CEA/TRICOM followed by i.t. boosting with rF-CEA/TRICOM. The results demonstrate that the antitumor activity induced by i.t. vaccination is superior to that induced by s.c. vaccination. For more advanced tumors, a s.c. priming vaccination, followed by i.t. boosting vaccinations was superior to either s.c. or i.t. vaccination alone. Both of these phenomena were observed in tumor models where the tumor-associated antigen is a foreign antigen and in a CEA-transgenic tumor model where the tumor-associated antigen is a self-antigen. The cytokine, granulocyte macrophage colony-stimulating factor admixed in vaccines, was shown to be essential in inducing the antitumor activity. These studies demonstrate that the diversified vaccine regimens that consisted of s.c. prime and i.t. boosts with CEA/TRICOM vectors could induce antitumor therapy superior to that seen by either route alone.